A major question in neural development is whether a common multipotential precursor generates neurons and macroglial cells of the CNS (central nervous system) or whether each cell type is derived from a separate population of precursor cells. Our hypothesis based on data from studies in different parts of the nervous system is that differentiation involves sequential restriction of an initially pluripotent population under specific environmental influences. To test this hypothesis we have developed a clonal culture assay for NEP (neuroepithelial) cells, from caudal neural tubes of El 0.5 rat embryos, which allows us to determine the molecular and cellular interactions that instruct pluripotent/ committed precursor cells to differentiate into neurons, astrocytes and oligodenrocytes. In this application we have proposed a series of in vitro experiments to study motoneuron differentiation from precursors. Specifically we plan the following experiments. I) Characterize and isolate the putative motoneuron precursors. Define the antigenic characteristics that specify motoneuron precursors and motoneurons and establish the relationship of the motoneuron precursor to the pluripotent precursor in mass and clonal culture. 2) Generate motoneuron precursor cell lines that recapitulate motoneuron development. Obtain immortalized neuroepithelial cell lines that differentiate into motoneurons and utilize them as a ready source of homogenous undifferentiated cells to study motoneuron differentiation. 3) Elucidate the developmental program that leads to the generation of motoneurons in vitro. Screen factors such as Sonic hedgehog CNTF/LIF, NT.3, and retinoic acid to identify molecules that promote motoneuron differentiation. Use cell lines and primary cultures to identify stages of development that are regulated by these factors. The results of the proposed experiments will provide critical insights into motoneuron development. Our experimental design will allow us to identify 1) The developmental relationship of motoneurons to other spinal cord cells 2) the identity of trophic and differentiation molecules that regulate normal motoneuron development. 3) The stage specific role of these identified molecules in regulating differentiation. Further, our results will provide an experimental framework to study other candidate molecules that may play a role in neuroepithelial development.